Calcium (Ca) release through the ryanodine receptor (RyR) is essential for regular heart contraction. Defects in RyR regulation cause imbalance in Ca homeostasis and contractile dysfunction in a variety of cardiac diseases. Since the most common cardiac pathologies (e.g. infarction, heart failure) are associated with oxidative stress, the main goal of this proposal is to define the molecular mechanisms of RyR dysfunction during oxidative stress. The RyR contains a large number of cysteine residues that can couple the cytosolic redox potential and Ca homeostasis. However, the functionally important redox-sensing sites on the RyR have not yet been identified. As a result, the molecular mechanisms of RyR dysfunction during oxidative stress remain largely unknown. This delays our progress in designing effective therapeutic interventions that can improve Ca homeostasis during cardiac diseases. Thus, more direct work identifying functionally important redox-sensing cysteines on RyR is essential to advance the field. We have recently discovered that oxidative stress activates the RyR by forming disulfide bonds between two neighboring subunits: intersubunit crosslinking. In this proposal we will test the hypothesis that intersubunit crosslinking is the mot functionally important redox modification of RyR responsible for the imbalance in Ca homeostasis during oxidative stress. This hypothesis will be tested using cutting-edge experimental techniques, such as RyR mutagenesis, single RyR channel recordings, and high resolution Ca imaging. In aim 1 we will identify specific cysteine residues on RyR that are involved in the crosslinking. Then, we will determine if mutation of these cysteines can maintain normal RyR function and Ca homeostasis during oxidative stress. In aim 2 we will define the molecular mechanisms of RyR dysfunction induced by the crosslinking. Calmodulin (CaM) bound to the RyR plays an important role in negative control of RyR activity. Our pilot studies suggest that the crosslinking causes dissociation of CaM from the RyR. Here, we will define if mutation of crosslinking cysteines can normalize Ca homeostasis during oxidative stress by preventing the CaM-RyR uncoupling. We will also explore whether stabilizing the CaM-RyR binding can protect the RyR function against oxidative stress in cardiomyocytes. By accomplishing these studies, we expect to define novel targets for future therapies that can improve Ca homeostasis during cardiac diseases associated with oxidative stress.